低聚木糖(xylooligosaccharides,XOS)是近年来备受关注的一种功能性低聚糖。XOS的甜度是蔗糖的30%～40%,与其他低聚糖,如低聚果糖(fructo oligosaccharide)和菊粉(inulin)相比,具有稳定性好、耐酸耐高温等优点[1]。除此之外,XOS不能被人体吸收,只能被肠道内的微生物利用,从而促进肠道内双歧杆菌(Bifidobacterium)、乳酸菌等有益菌产生对宿主有益的小分子脂肪酸,选择性抑制大肠杆菌(Escherichia coli)、梭状芽孢杆菌(Clostridium)等有害菌和致病菌的生长,因此XOS被认为是具有益生元活性的可溶性膳食纤维[2-4],已被广泛应用于食品、保健品或者饲料添加剂中，且可作为糖尿病患者食品的增味剂。
XOS天然存在于水果、蔬菜中,也可以通过水解木聚糖(xylan)生产。XOS是由木糖残基链组成的寡糖,一般由2～7个木糖分子聚合而成,主链通过β-1,4-糖苷键连接,有不同的侧基修饰形成支链结构,不同的木聚糖来源和不同的工艺生产出的XOS的聚合度(degree of polymerization,DP)、侧基以及侧基的取代方式差别很大[5-7]。DP为2～4的XOS具有更高的益生元活性,也因此更受人们青睐[8]。本文对近年来XOS的生产技术研究进展进行了总结,并介绍了XOS作为饲料添加剂对动物的益生作用,以期为XOS的工业化生产和应用提供依据。
1 低聚木糖生产原料
木质纤维素作为农林业废弃物,其主要由纤维素(cellulose)、半纤维素(hemicellulose)和木质素(lignin)3种组分组成。XOS可通过木聚糖水解的方法获得,而木聚糖是木质纤维素中半纤维素的主要成分,因此,在工业生产中大多以农业残余物作为原料生产具有高附加值的XOS。表1列举了几种常见木质纤维素原料的组成,其中木聚糖含量在15%～31%,原料的木聚糖含量越高,越有利于低成本地生产XOS,其中玉米芯、甘蔗渣、小麦秸秆等来源广泛,木聚糖含量相对较高,是XOS工业生产中普遍使用的原料。
表1 木质纤维素原料的组成成分
Table 1 Composition of lignocellulosic materials
材料纤维素/%木聚糖/%木质素/%参考文献玉米芯32.931.512.5[9]甘蔗渣39.823.622.8[10]小麦秸秆34.922.821.4[9]松木38.820.731.2[11]水曲柳37.020.629.5[11]玉米秸秆30.320.019.6[9]芒草35.919.519.6[12]油茶壳13.419.539.1[13]杨木40.416.926.2[9]杉木48.115.034.1[11]
2 低聚木糖生产技术研究进展
由于木质纤维素中木质素、果胶(pectin)等物质会影响木聚糖的提取率,因此需要打破木质纤维素的致密结构,使更多的半纤维素暴露出来,从而提高XOS的提取效率[14]。XOS可以采用物理、化学方法直接生产,也可以在物理、化学预处理技术的基础上结合木聚糖酶酶解生产。表2和表3总结了近年来文献中报道的XOS生产技术。
2.1 物理、化学生产法
2.1.1 自水解法
在高温高压的环境中,木质纤维素中半纤维素发生水解断裂,使木聚糖发生自身水解反应释放出XOS和木糖[15]。SUREK等[16]发现自水解的温度和保温时间对产物产量有极大影响,在190 ℃、保温5 min的条件下处理榛子(Corylus avellana L)壳,XOS收率达到原料木聚糖的62%。要想使低DP的XOS的百分比达到最大,在此温度下则需要30 min的保温时间,并且木糖、乙酸和糠醛浓度随处理强度的增加而增加。ZHANG等[17]在200 ℃的条件下对甘蔗渣进行自水解,在10 min内得到的XOS产率为50.35%,若想生产低DP的XOS则需要更长的反应时间,而随着反应时间的增加,大量的木聚糖和XOS被降解为木糖和副产物。反应时间的延长造成了副产物的大量产生,不仅不能保证XOS的产率,还对XOS的纯度造成影响。
表2 化学/物理法生产XOS
Table 2 XOS production using chemical/physical methods
原料处理方法产率参考文献自水解法甘蔗渣200℃,10 min50.35%[17]榛子壳190 ℃,5 min62.0%[16]芦苇屑170 ℃,30 min49.5%[28]酸处理法玉米芯pH 2.7 乙酸,150 ℃,30 min45.91%[18]葡萄渣8.4% NaOH,固液比1∶18(g∶mL),120℃,90 min96.28%[26]葡萄渣0.8% H2SO4,固液比1∶18(g∶mL),120℃,90 min74.58%[26]杨木φ(乙酸)=5%,170℃,30 min55.8%[31]甘蔗渣ω(乙酸)=10%,150℃,45 min39.1%[32]玉米芯φ(木糖酸)=5%,145 ℃,75 min51.24%[9]玉米秸秆φ(木糖酸)=25%,157 ℃,35 min23.11%[9]小麦秸秆φ(木糖酸)=5%,153 ℃,75 min35.6%[9]杨木屑φ(木糖酸)=5%,167 ℃,35 min30.23%[9]甘蔗渣0.64 mol/L 木糖酸,154 ℃,42 min44.5%[10]无机盐法油茶壳ω(ZnCl2)=0.5%,170℃,30 min61.38%[19]甘蔗渣0.1 mol/L MgCl2,180 ℃,10 min53.79%[20]甘蔗渣0.1 mol/L FeCl2,140 ℃,30 min41.89%[20]甘蔗渣0.05 mol/L FeCl2 + 0.05 mol/L MgCl2,140 ℃,30 min54.68%[20]其他处理法甘蔗渣0.24 mol/L H2SO4微波31 min290.2 mg/g[25]桉木1% Ca (OH) 2,0.8%活性炭,1.4% H2SO4DP 2～4含量为9.25 g/L[33]油棕榈皮亚临界H2O-CO2,180 ℃,60 min81.6 mg /g[34]甘蔗渣ω(H2SO4)=0.5%,190 ℃,5 min爆破40%左右[24]芒草15 bar,200 ℃,10 min爆破52%[23]
2.1.2 酸处理法
酸处理的目的是提高半纤维素水解程度,从而提高XOS的产率。有研究用玉米芯生产XOS和可发酵糖,使用乙酸、HCl和H2SO4三种不同的酸在pH 2.7、150 ℃、30 min的相同条件下对玉米芯进行处理,在所选择的酸中,乙酸处理XOS的收率最高达到45.91%,与另外2种酸相比,经过乙酸处理的玉米芯暴露出更大的表面积,而且更粗糙,更有利于后续的纤维素酶解[18]。GUO等[9]在不同温度和时间下用自生产的木糖酸共同生产XOS和葡萄糖。通过实验发现玉米芯在145 ℃反应75 min时XOS收率最高为54.16%,并且纤维素酶解效率可达到100%。
2.1.3 无机盐法
与酸处理相比,无机盐溶液处理对设备的腐蚀性低,且有更高的催化活性。无机盐处理主要利用了路易斯酸(Lewis acid)和酸碱质子理论(Brønsted-Lowry acid-base theory)的特性,在水中添加无机盐发生水解反应,金属离子和水形成复杂的络合物,这些配位化合物和水中的氢键可以帮助半纤维素降解。YOU等[19]使用ZnCl2催化活化油茶壳生产DP为2～5的XOS,发现在质量分数0.5% ZnCl2催化下于170 ℃ 蒸煮30 min时XOS的产率最大达到61.38%。ZHANG等[20]在140 ℃下用MgCl2和FeCl2共催化甘蔗渣30 min,得到54.68%的XOS,其中有29.34%的木二糖(xylobiose)和20.94%的木三糖(xylotriose),并且发现无机盐共催化比单种无机盐处理增加了XOS的总产量,并且提高了木二糖和木三糖的产量。
2.1.4 其他处理法
除了上述方法之外,还有蒸汽爆破法、微波处理法等生产XOS的方法。蒸汽爆破对环境污染小成本低、处理时间短,在蒸汽爆破期间,将植物生物质进料到爆破容器中通入蒸汽,使木质纤维素经受一段时间的高压和高温,在高温高压的环境中,木质纤维素中半纤维素发生水解破裂,然后快速减压使物料的结构变得分散,实现木质纤维素各个部分的有效分离[15,21-22]。
BHATIA等[23]通过实验发现蒸汽爆破预处理条件为200 ℃,保温10 min,芒草爆破产物中最高XOS收率达到52%。CARVALHO等[24]使用质量分数 0.5% H2SO4对甘蔗渣进行过夜处理,然后190 ℃保温5 min爆破,XOS的得率约为40%。
微波处理法同样是一种绿色高效的生产方法,有能耗低、传热均匀、反应即关即停等优点。BIAN等[25]用微波辅助酸水解的方法从甘蔗渣的半纤维素组分中制备XOS,当微波加热至90 ℃时，用0.24 mol/L的H2SO4水解31 min,可得到290.2 mg/g的XOS。
2.2 物理/化学-酶解联合生产法
酶解法生产XOS是一种反应条件温和,更适合工业生产的方法。COSTA等[26]使用H2SO4和NaOH两种溶剂与酶解法做了比较,证明了酶混合物可作为传统方法的一种替代方法,使用黑曲霉(Aspergillus niger)3T5B8产生的木聚糖酶对葡萄皮渣粉直接进行酶解,得到的XOS收率高达88.68%。为了更好地发挥木聚糖酶的作用,更多研究者在酶解前会先用预处理的方式使半纤维素暴露。碱预处理的主要作用是去除木质素以及果胶、脂肪、蛋白质等。将木质素溶解除去,然后可以对留下的纤维素和半纤维素进行处理从而得到糖单体和低聚物[27]。研究发现，与酸处理和水热处理相比，碱性预处理能更好地去除木质素并暴露出更多木聚糖酶的作用位点，促进后续的酶解过程[28]。SINGH等[29]优化了两步碱预处理工艺,槟榔皮在质量分数为10%的NaOH溶液中于65 ℃条件下孵育8 h,再经过水热处理(121 ℃持续1 h)的最佳条件下,经过酶水解后产生35 g/100 g纯木聚糖的XOS。HAO等[30]利用过氧化氢-乙酸(HPAC)并以H2SO4为催化剂预处理杨木,通过两步木聚糖酶和纤维素酶处理将固体残留物用于生产XOS和葡萄糖,用添加了50 mmol/L H2SO4作为催化剂的30%H2O2和99%乙酸预处理2 h,酶解后XOS的产率可以达到16.9 g/kg杨木。
表3 化学/物理预处理-酶解联合生产XOS
Table 3 XOS production using chemical/physical pretreatment and enzymatic hydrolysis
原料预处理方法酶解条件产量参考文献直接酶解法葡萄渣无pH5.0,40℃,4 h88.68%[26]化学预处理-酶解啤酒糟0.7% Na2CO3,0.1% H2O2,115 ℃,30 minpH 5.5, 48 ℃,3 h23.87 mg/g啤酒糟[35]甘蔗渣10% KOH,2.36% H2O2,50 ℃,6 hpH 5.0,36 ℃,24 h43%[36]槟榔皮10% NaOH,65 ℃,8 h,121 ℃,1 hpH 4,50 ℃,24 h35 g/100 g木聚糖[29]甘蔗渣10% NaOH,1.5% H2O2,70 ℃,6 hpH 5.0,50 ℃逐步酶水解1.78 g/L[37]杨木60% HPAC,50 mmol/L H2SO4,2 hpH 5.0,50 ℃,24 h16.9 g/kg[30]杨木60% HPAC,200 mmol/L H2SO4,2 hpH 5.0,50 ℃,24 h14.2 g/kg[30]稻壳pH 5.0柠檬酸缓冲液,0.9 MPa蒸煮50 minpH 6.0,50 ℃,3 h8.26 mg/g[38]其他预处理-酶解稻壳微波功率640 W,3.2 min,pH 5.0柠檬酸缓冲液pH 5.0,59 ℃,3 h4.94 mg/g[39]玉米芯196 ℃,5 minpH 7.0,70 ℃,2.5 h28.6 g/100 g[40]小麦秆200 ℃,加热50 s,保温4 minpH 4.8,50 ℃8.9 g/100 g[41]
3 低聚木糖对动物的益生作用研究进展
3.1 改变肠道菌群的组成和代谢活性
XOS可以改变肠道微生物的组成,增加益生菌的数量,产生有利于健康的脂肪酸。PAN等[42]探讨了日粮XOS对不同年龄猪的肠道微生物组成和活性的影响,发现在育肥期添加XOS会增加猪肠道内容物中乙酸、直链脂肪酸和短链脂肪酸的浓度,改变肠道菌群的组成和代谢活性。
3.2 改善肠道屏障,增强免疫功能
肠道屏障可以保护宿主不被致病菌危害,肠道微生物对宿主的免疫力有很大影响。有研究通过用XOS和对照饮食喂养小鼠进行了肠道微生物评估,证明XOS可以改变肠道微生物群,改善肠道屏障,加强肠道致糖尿病抗原的控制[43]。EAIMWORAWU-THIKUL等[44]在雄性Wistar大鼠上证明了XOS可以减少全身性炎症,增加小梁厚度,减少破骨细胞和活跃的糜烂面,并恢复了矿物质沉积和骨形成速率。YIN等[45]发现膳食补充XOS对生长性能、血细胞、生化参数以及肠道形态几乎没有影响，但是XOS喂养的仔猪炎症状态和肠道屏障得到改善。菌群分析表明,XOS影响了乳杆菌属(Lactobacillus)、链球菌属(Streptococcus)和Turicibacter的相对丰度。微生物的改变可能进一步涉及碳水化合物的代谢、细胞运动、细胞过程和信号传导、脂质代谢以及其他氨基酸的代谢。
3.3 增加动物日重,提高饲料效率
由于应激反应的影响,在动物饲养中很容易出现料肉比(feed conversion ratio,FCR)过高的现象,XOS可以降低FCR,增加动物日增质量。RIBEIRO等[46]研究了XOS对肉鸡性能的影响,显示掺入XOS的饲喂或外源β-1,4-木聚糖酶的膳食补充均增加了小麦日粮的营养价值,动物性能的改善伴随着在胃肠道上部定居的微生物种群的转移。
研究发现在饲料中添加XOS喂养的猪具有更高的平均日增重和饲料效率,XOS提高了胰蛋白酶活性,降低粪便中NH3浓度以及粪便大肠杆菌的数量,增加了乳杆菌的数量[47]。ABDELMALEK等[48]发现在饲料中添加XOS喂养欧洲鲈鱼(Dicentrarchus labrax),可显著增加体重,增加蛋白质效率比和饲料转化率,改善生长并刺激免疫力,同时增强抗感染能力。
3.4 改善抗氧化能力
ABASUBONG等[49]研究木寡糖对钝嘴鲷(Megalobrama amblycephala)的生长性能、抗氧化能力和针对嗜水气单胞菌的免疫反应的影响,研究表明,饮食中添加质量分数1.5%XOS可以显著改善钝嘴鲷的生长性能、抗氧化能力、先天免疫力和嗜水杆菌抗性。YU等[50]的数据证明了XOS可增加小鼠粪便中的乳杆菌属和双歧杆菌属含量,降低肠球菌属、肠杆菌属和梭状芽胞杆菌属含量,体外抗氧化结果表明,XOS与植物乳杆菌(Lactobacillus plantarum)均具有自由基清除活性,且联合使用具有更好的抗氧化活性。
4 结语
XOS以其良好的稳定性,独特的生理特性获得了人们的青睐。XOS作为饲料添加剂能够改善猪的肠道菌群,促进益生菌生长,依靠肠道屏障减少炎症,起到预防疾病的效果,还能增加动物的日重,提高蛋鸡的生产性能,在水产养殖中也能起到一定作用。随着人们对XOS功能的探索,XOS的应用会越来越广泛。值得注意的是,XOS的制备工艺至今还未成熟,如何更低成本、低污染、低能耗地生产XOS已经成为研究的热点,而且XOS从生产底物中提纯的成本过高,如何简单快捷、低成本地分离XOS也将是未来研究的热点。
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